1. Field of the Invention
The present invention relates to the genetic transformation of sugarbeet plants. Specifically, the present invention is directed to a genotype-independent transformation of sugarbeet plants. The present invention utilizes bombardment techniques in the transformation.
2. Background of the Invention and Related Information
Sugarbeet is economically the most important sucrose-producing crop in temperate regions of the world and provides about 37% of the world's sugar. In addition, the sugarbeet plant is useful in the production of many other commercially important carbohydrates.
In contrast to many other plant species, sugarbeet has proven recalcitrant to regeneration after transformation. Some plants are totipotent, in that entire plants can be regenerated from a single plant cell, and transgenic plants can, therefore, be engineered from genetically engineered plant cells. However, many reports have demonstrated that the regeneration of sugarbeet after transformation is difficult to reproduce, is unpredictable and is strongly genotype dependent.
A number of effective DNA-delivery systems are available for the transfer of foreign genes into plant genomes. Effective gene transfer into sugarbeet using the gram-negative soil bacterium Agrobacterium has been demonstrated. However, Agrobacterium-based transformation of sugarbeet is confined to relatively few genotypes. Regeneration from tissue culture following Agrobacterium infection has also proved difficult with somaclonal variation resulting from tissue-culture induced mutations. Several patent applications are directed to the subject of Agrobacterium-mediated transformation of sugarbeets, including U.S. Pat. No. 5,614,395 to RYALS et al., PCT International Application PCT/DK92/00108 to MIKKELSEN et al., and World Intellectual Property Organization Application No. WO 93/07272 to LETHAM. RYALS et al., MIKKELSEN et al., and LETHAM, are hereby expressly incorporated by reference as though set forth in full herein.
A number of studies have indicated that Agrobacterium-mediated transformation may result in two problems which need to be addressed prior to the release of any genetically engineered plant into the environment. First, molecular analysis of genomic DNA from plants engineered in this manner has suggested the presence of vector sequences outside the transferred DNA borders (RAMANATHAN et al., Plant Mol. Biol. 28, pp. 1149-1154 (1995)). In addition, the Agrobacterium microbe has been found to persist on the surface and within tissues of soil-grown transformed plants up to 12 months following transformation (MATZIK et al., Mol. Plant-Microbe Interact. 9, pp. 373-381 (1996)). These two problems make the use of Agrobacterium in sugarbeet transformation considerably less attractive.
Other methods of transforming plants include directly transferring DNA by using electroporation or chemical fusagens such as polyethylene glycol with calcium phosphate. World Intellectual Property Organization Application No. WO 91/00358 to J.O slashed.RSBOE teaches a method for introducing genetic material into plant cells such as tobacco and sugarbeet using ultrasound treatment. Each of these methods, however, requires the use of protoplasts, and as noted above, regeneration of sugarbeet plants from single cells has proven to be very difficult. Even in cases in which regeneration from single cells has been reported, genotype specificities can severely hinder the transformation and regeneration of these crops. DNA can also be effectively transferred via the mechanical introduction of plasmid DNA via microinjection. Although this method is not subject to host-range limitations, regeneration from single cells is a requirement. In view of the foregoing, there is a need for a method to stably introduce DNA into sugarbeet plant cells without having to regenerate from single cells.
Particle bombardment is a technique that has proven effective in the genetic transformation of other species of plant. This technique utilizes very small particles which are delivered at a high velocity. The particles act as carriers of biologically active DNA into germline plant cells, tissues, and organs. Particle bombardment has been successfully employed for the transformation of soybean (U.S. Pat. Nos. 5,015,580 and 5,503,998 to CHRISTOU et al., the entire contents of which are expressly incorporated by reference as though set forth in full herein) and other crops refractory to alternative gene transfer methods. MAHN et al. demonstrated transient gene expression using particle bombardment of sugarbeet in J. Experimental Botany 46 (291), pp 1625-1628 (1995), the entire content of which is hereby incorporated by reference as though set forth in full herein. However, to date, particle bombardment has not successfully been used to create a stably transformed sugarbeet.